RESUMEN
BACKGROUND: Relapsed and refractory acute myeloid leukemia (AML) carries a dismal prognosis. CAR T cells have shown limited efficacy in AML, partially due to dysfunctional autologous T cells and the extended time for generation of patient specific CAR T cells. Allogeneic NK cell therapy is a promising alternative, but strategies to enhance efficacy and persistence may be necessary. Proteasome inhibitors (PI) induce changes in the surface proteome which may render malignant cells more vulnerable to NK mediated cytotoxicity. Here, we investigated the potential benefit of combining PIs with CAR-expressing allogeneic NK cells against AML. METHODS: We established the IC50 concentrations for Bortezomib and Carfilzomib against several AML cell lines. Surface expression of class-I HLA molecules and stress-associated proteins upon treatment with proteasome inhibitors was determined by multiparameter flow cytometry. Using functional in vitro assays, we explored the therapeutic synergy between pre-treatment with PIs and the anti-leukemic efficacy of NK cells with or without expression of AML-specific CAR constructs against AML cell lines and primary patient samples. Also, we investigated the tolerability and efficacy of a single PI application strategy followed by (CAR-) NK cell infusion in two different murine xenograft models of AML. RESULTS: AML cell lines and primary AML patient samples were susceptible to Bortezomib and Carfilzomib mediated cytotoxicity. Conditioned resistance to Azacitidine/Venetoclax did not confer primary resistance to PIs. Treating AML cells with PIs reduced the surface expression of class-I HLA molecules on AML cells in a time-and-dose dependent manner. Stress-associated proteins were upregulated on the transcriptional level and on the cell surface. NK cell mediated killing of AML cells was enhanced in a synergistic manner. PI pre-treatment increased effector-target cell conjugate formation and Interferon-γ secretion, resulting in enhanced NK cell activity against AML cell lines and primary samples in vitro. Expression of CD33- and CD70-specific CARs further improved the antileukemic efficacy. In vivo, Bortezomib pre-treatment followed by CAR-NK cell infusion reduced AML growth, leading to prolonged overall survival. CONCLUSIONS: PIs enhance the anti-leukemic efficacy of CAR-expressing allogeneic NK cells against AML in vitro and in vivo, warranting further exploration of this combinatorial treatment within early phase clinical trials.
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Bortezomib , Células Asesinas Naturales , Leucemia Mieloide Aguda , Inhibidores de Proteasoma , Receptores Quiméricos de Antígenos , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/efectos de los fármacos , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Receptores Quiméricos de Antígenos/inmunología , Animales , Ratones , Línea Celular Tumoral , Bortezomib/farmacología , Bortezomib/uso terapéutico , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Inmunoterapia Adoptiva/métodos , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Endogámicos NOD , Ratones SCID , FemeninoRESUMEN
Immunoproteasome is a specialized form of proteasome which plays a crucial role in antigen processing and presentation, and enhances immune responses against malignant cells. This review explores the role of immunoproteasome in the anti-tumor immune responses, including immune surveillance and modulation of the tumor microenvironment, as well as its potential as a target for cancer immunotherapy. Furthermore, we have also discussed the therapeutic potential of immunoproteasome inhibitors, strategies to enhance antigen presentation and combination therapies. The ongoing trials and case studies in urology, melanoma, lung, colorectal, and breast cancers have also been summarized. Finally, the challenges facing clinical translation of immunoproteasome-targeted therapies, such as toxicity and resistance mechanisms, and the future research directions have been addressed. This review underscores the significance of targeting the immunoproteasome in combination with other immunotherapies for solid tumors and its potential broader applications in other diseases.
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Inmunoterapia , Neoplasias , Complejo de la Endopetidasa Proteasomal , Microambiente Tumoral , Humanos , Neoplasias/inmunología , Neoplasias/terapia , Inmunoterapia/métodos , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/inmunología , Microambiente Tumoral/inmunología , Animales , Inhibidores de Proteasoma/uso terapéutico , Inhibidores de Proteasoma/farmacología , Carcinoma/inmunología , Carcinoma/terapiaRESUMEN
TRIM44, a tripartite motif (TRIM) family member, is pivotal in linking the ubiquitin-proteasome system (UPS) to autophagy in multiple myeloma (MM). However, its prognostic impact and therapeutic potential remain underexplored. Here, we report that TRIM44 overexpression is associated with poor prognosis in a Multiple Myeloma Research Foundation (MMRF) cohort of 858 patients, persisting across primary and recurrent MM cases. TRIM44 expression notably increases in advanced MM stages, indicating its potential role in disease progression. Single-cell RNA sequencing across MM stages showed significant TRIM44 upregulation in smoldering MM (SMM) and MM compared to normal bone marrow, especially in patients with t(4;14) cytogenetic abnormalities. This analysis further identified high TRIM44 expression as predictive of lower responsiveness to proteasome inhibitor (PI) treatments, underscoring its critical function in the unfolded protein response (UPR) in TRIM44-high MM cells. Our findings also demonstrate that TRIM44 facilitates SQSTM1 oligomerization under oxidative stress, essential for its phosphorylation and subsequent autophagic degradation. This process supports the survival of PI-resistant MM cells by activating the NRF2 pathway, which is crucial for oxidative stress response and, potentially, other chemotherapy-induced stressors. Additionally, TRIM44 counters the TRIM21-mediated suppression of the antioxidant response, enhancing MM cell survival under oxidative stress. Collectively, our discoveries highlight TRIM44's significant role in MM progression and resistance to therapy, suggesting its potential value as a therapeutic target.
Asunto(s)
Mieloma Múltiple , Complejo de la Endopetidasa Proteasomal , Proteínas de Motivos Tripartitos , Mieloma Múltiple/patología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/genética , Humanos , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Pronóstico , Línea Celular Tumoral , Complejo de la Endopetidasa Proteasomal/metabolismo , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Autofagia/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Inhibidores de Proteasoma/farmacología , Resistencia a Antineoplásicos/genética , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
In this review we explore innovative approaches in the treatment of hematologic cancers by combining various therapeutic modalities. We discuss the synergistic potential of combining inhibitors targeting different cellular pathways with immunotherapies, molecular therapies, and hormonal therapies. Examples include combining PI3K inhibitors with proteasome inhibitors, NF-κB inhibitors with immunotherapy checkpoint inhibitors, and neddylation inhibitors with therapies targeting the tumor microenvironment. Additionally, we discuss the potential use of small molecules and peptide inhibitors in hematologic cancer treatment. These multidimensional therapeutic combinations present promising strategies for enhancing treatment efficacy and overcoming resistance mechanisms. However, further clinical research is required to validate their effectiveness and safety profiles in hematologic cancer patients.
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Neoplasias Hematológicas , Humanos , Neoplasias Hematológicas/tratamiento farmacológico , Inmunoterapia/métodos , Terapia Molecular Dirigida , Microambiente Tumoral/efectos de los fármacos , Animales , Inhibidores de Proteasoma/uso terapéutico , Inhibidores de Proteasoma/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacologíaRESUMEN
Proteasome inhibitors have been employed in the treatment of relapsed multiple myeloma and mantle cell lymphoma. The observed toxicity caused by proteasome inhibitors is a universal phenotype in numerous cancer cells with different sensitivity. In this study, we investigate the conserved mechanisms underlying the toxicity of the proteasome inhibitor bortezomib using gene editing approaches. Our findings utilizing different caspase knocking out cells reveal that bortezomib induces classic intrinsic apoptosis by activating caspase-9 and caspase-3/7, leading to pore-forming protein GSDME cleavage and subsequent lytic cell death or called secondary necrosis, a phenotype also observed in many apoptosis triggers like TNFα plus CHX, DTT and tunicamycin treatment in HeLa cells. Furthermore, through knocking out of nearly all BH3-only proteins including BIM, BAD, BID, BMF and PUMA, we demonstrate that NOXA is the sole BH3-only protein responsible for bortezomib-induced apoptosis. Of note, NOXA is well known for selectively binding to MCL-1 and A1, but our studies utilizing different BH3 mimetics as well as immunoprecipitation assays indicate that, except for the constitutive interaction of NOXA with MCL-1, the accumulation of NOXA after bortezomib treatment allows it to interact with BCL-XL, then simultaneous relieving suppression on apoptosis by both anti-apoptotic proteins BCL-XL and MCL-1. In addition, though bortezomib-induced significant ER stress and JNK activation were observed in the study, further genetic depletion experiments prove that bortezomib-induced apoptosis occurs independently of ER stress-related apoptosis factor CHOP and JNK. In summary, these results provide a solid conclusion about the critical role of NOXA in inactivation of BCL-XL except MCL-1 in bortezomib-induced apoptosis.
Asunto(s)
Apoptosis , Bortezomib , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Inhibidores de Proteasoma , Proteínas Proto-Oncogénicas c-bcl-2 , Proteína bcl-X , Humanos , Apoptosis/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Bortezomib/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína bcl-X/metabolismo , Proteína bcl-X/genética , Inhibidores de Proteasoma/farmacología , Factor de Transcripción CHOP/metabolismo , Factor de Transcripción CHOP/genética , Células HeLa , Sistema de Señalización de MAP Quinasas/efectos de los fármacosRESUMEN
Proteasome inhibitors (PIs), bortezomib, carfilzomib, and ixazomib, are the first-line treatment for multiple myeloma (MM). They inhibit cytosolic protein degradation in cells, which leads to the accumulation of misfolded and malfunctioned proteins in the cytosol and endoplasmic reticulum, resulting in cell death. Despite being a breakthrough in MM therapy, malignant cells develop resistance to PIs via different mechanisms. Understanding these mechanisms drives research toward new anticancer agents to overcome PI resistance. In this review, we summarize the mechanism of action of PIs and how MM cells adapt to these drugs to develop resistance. Finally, we explore these mechanisms to present strategies to interfere with PI resistance. The strategies include new inhibitors of the ubiquitin-proteasome system, drug efflux inhibitors, autophagy disruption, targeting stress response mechanisms, affecting survival and cell cycle regulators, bone marrow microenvironment modulation, and immunotherapy. We list potential pharmacological targets examined in in vitro, in vivo, and clinical studies. Some of these strategies have already provided clinicians with new anti-MM medications, such as panobinostat and selinexor. We hope that further exploration of the subject will broaden the range of therapeutic options and improve patient outcomes.
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Resistencia a Antineoplásicos , Mieloma Múltiple , Inhibidores de Proteasoma , Humanos , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Complejo de la Endopetidasa Proteasomal/metabolismo , Autofagia/efectos de los fármacosRESUMEN
The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit ß5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes.
Asunto(s)
Mesotelioma , Complejo de la Endopetidasa Proteasomal , Inhibidores de Proteasoma , Proteínas WT1 , Humanos , Línea Celular Tumoral , Proteínas WT1/inmunología , Inhibidores de Proteasoma/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/inmunología , Mesotelioma/inmunología , Mesotelioma/tratamiento farmacológico , Epítopos/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Antígeno HLA-A24/inmunología , Mesotelioma Maligno/inmunología , Mesotelioma Maligno/tratamiento farmacológico , Epítopos de Linfocito T/inmunología , OligopéptidosRESUMEN
Because hepatic stellate cells (HSCs) play a major role in fibrosis, we focused on HSCs as a potential target for the treatment of liver fibrosis. In this study, we attempted to identify drug candidates to inactivate HSCs and found that several proteasome inhibitors (PIs) reduced HSC viability. Our data showed that a second-generation PI, carfilzomib (CZM), suppressed the expression of fibrotic markers in primary murine HSCs at low concentrations of 5 or 10 nM. Since CZM was not toxic to HSCs up to a concentration of 12.5 nM, we examined its antifibrotic effects further. CZM achieved a clear reduction in liver fibrosis in the carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis without worsening of liver injury. Mechanistically, RNA sequence analysis of primary HSCs revealed that CZM inhibits mitosis in HSCs. In the CCl4-injured liver, amphiregulin, which is known to activate mitogenic signaling pathways and fibrogenic activity and is upregulated in murine and human metabolic dysfunction-associated steatohepatitis (MASH), was downregulated by CZM administration, leading to inhibition of mitosis in HSCs. Thus, CZM and next-generation PIs in development could be potential therapeutic agents for the treatment of liver fibrosis via inactivation of HSCs without liver injury.
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Células Estrelladas Hepáticas , Cirrosis Hepática , Oligopéptidos , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Animales , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/inducido químicamente , Ratones , Masculino , Modelos Animales de Enfermedad , Tetracloruro de Carbono , Humanos , Ratones Endogámicos C57BL , Mitosis/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Anfirregulina/metabolismo , Supervivencia Celular/efectos de los fármacosRESUMEN
OBJECTIVE: Proteasomes are conserved proteases crucial for proteostasis in eukaryotes and are promising drug targets for protozoan parasites. Yet, the proteasomes of Entamoeba histolytica remain understudied. The study's objective was to analyse the differences in the substrate binding pockets of amoeba proteasomes from those of host, and computational modelling of ß5 catalytic subunit, with the goal of finding selective inhibitors. RESULTS: Comparative sequence analysis revealed differences in substrate binding sites of E. histolytica proteasomes, especially in the S1 and S3 pockets of the catalytic beta subunits, implying differences in substrate preference and susceptibility to inhibitors from host proteasomes. This was strongly supported by significantly lower sensitivity to MG132 mediated inhibition of amoebic proteasome ß5 subunit's chymotryptic activity compared to human proteasomes, also reflected in lower sensitivity of E. histolytica to MG132 for inhibition of proliferation. Computational models of ß4 and ß5 subunits, and a docked ß4-ß5 model revealed a binding pocket between ß4-ß5, similar to that of Leishmania tarentolae. Selective inhibitors for visceral leishmaniasis, LXE408 and compound 8, docked well to this pocket. This functional and sequence-based analysis predicts differences between amoebic and host proteasomes that can be utilized to develop rationally designed, selective inhibitors against E. histolytica.
Asunto(s)
Entamoeba histolytica , Complejo de la Endopetidasa Proteasomal , Entamoeba histolytica/enzimología , Entamoeba histolytica/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Humanos , Sitios de Unión , Leupeptinas/farmacología , Especificidad por Sustrato , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Inhibidores de Proteasoma/farmacología , Simulación del Acoplamiento Molecular , Secuencia de Aminoácidos , Dominio Catalítico , Unión Proteica , Modelos MolecularesRESUMEN
Proteasome inhibitors (PIs), such as bortezomib and calfizomib, were backbone agents in the treatment of multiple myeloma (MM). In this study, we investigated bortezomib interactors in MM cells and identified dihydrolipoamide dehydrogenase (DLD) as a molecular target of bortezomib. DLD catalyzes the oxidation of dihydrolipoamide to form lipoamide, a reaction that also generates NADH. Our data showed that bortezomib bound to DLD and inhibited DLD's enzymatic function in MM cells. DLD knocked down MM cells (DLD-KD) had decreased levels of NADH. Reduced NADH suppressed assembly of proteasome complex in cells. As a result, DLD-KD MM cells had decreased basal-level proteasome activity and were more sensitive to bortezomib. Since PIs were used in many anti-MM regimens in clinics, we found that high expression of DLD correlated with inferior prognosis of MM. Considering the regulatory role of DLD in proteasome assembly, we evaluated DLD targeting therapy in MM cells. DLD inhibitor CPI-613 showed a synergistic anti-MM effect with bortezomib in vitro and in vivo. Overall, our findings elucidated DLD as an alternative molecular target of bortezomib in MM. DLD-targeting might increase MM sensitivity to PIs.
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Bortezomib , Dihidrolipoamida Deshidrogenasa , Mieloma Múltiple , Bortezomib/farmacología , Humanos , Dihidrolipoamida Deshidrogenasa/metabolismo , Dihidrolipoamida Deshidrogenasa/genética , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/enzimología , Animales , Línea Celular Tumoral , Complejo de la Endopetidasa Proteasomal/metabolismo , Antineoplásicos/farmacología , Ratones , Inhibidores de Proteasoma/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , NAD/metabolismo , Femenino , Masculino , Terapia Molecular DirigidaRESUMEN
Computational pharmacogenomics can potentially identify new indications for already approved drugs and pinpoint compounds with similar mechanism-of-action. Here, we used an integrated drug repositioning approach based on transcriptomics data and structure-based virtual screening to identify compounds with gene signatures similar to three known proteasome inhibitors (PIs; bortezomib, MG-132, and MLN-2238). In vitro validation of candidate compounds was then performed to assess proteasomal proteolytic activity, accumulation of ubiquitinated proteins, cell viability, and drug-induced expression in A375 melanoma and MCF7 breast cancer cells. Using this approach, we identified six compounds with PI properties ((-)-kinetin-riboside, manumycin-A, puromycin dihydrochloride, resistomycin, tegaserod maleate, and thapsigargin). Although the docking scores pinpointed their ability to bind to the ß5 subunit, our in vitro study revealed that these compounds inhibited the ß1, ß2, and ß5 catalytic sites to some extent. As shown with bortezomib, only manumycin-A, puromycin dihydrochloride, and tegaserod maleate resulted in excessive accumulation of ubiquitinated proteins and elevated HMOX1 expression. Taken together, our integrated drug repositioning approach and subsequent in vitro validation studies identified six compounds demonstrating properties similar to proteasome inhibitors.
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Bortezomib , Reposicionamiento de Medicamentos , Inhibidores de Proteasoma , Humanos , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/química , Reposicionamiento de Medicamentos/métodos , Bortezomib/farmacología , Transcriptoma , Complejo de la Endopetidasa Proteasomal/metabolismo , Línea Celular Tumoral , Células MCF-7 , Simulación del Acoplamiento Molecular , Antineoplásicos/farmacología , Antineoplásicos/química , Puromicina/farmacología , Perfilación de la Expresión Génica , Supervivencia Celular/efectos de los fármacosRESUMEN
Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf_proteasome ß5 active-site (Pfß5). A potent analog (SW584) showed efficacy in a mouse model of human malaria after oral dosing. SW584 had a low propensity to generate resistance (minimum inoculum for resistance [MIR] >109) and was synergistic with dihydroartemisinin. Pf_proteasome purification was facilitated by His8-tag introduction onto ß7. Inhibition of Pfß5 correlated with parasite killing, without inhibiting human proteasome isoforms or showing cytotoxicity. The Pf_proteasome_SW584 cryoelectron microscopy (cryo-EM) structure showed that SW584 bound non-covalently distal from the catalytic threonine, in an unexplored pocket at the ß5/ß6/ß3 subunit interface that has species differences between Pf and human proteasomes. Identification of a reversible, species selective, orally active series with low resistance propensity provides a path for drugging this essential target.
Asunto(s)
Antimaláricos , Piperidinas , Plasmodium falciparum , Inhibidores de Proteasoma , Piperidinas/química , Piperidinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Animales , Antimaláricos/farmacología , Antimaláricos/química , Humanos , Ratones , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/síntesis química , Administración Oral , Complejo de la Endopetidasa Proteasomal/metabolismo , Malaria/tratamiento farmacológico , Malaria/parasitología , Amidas/química , Amidas/farmacología , Amidas/síntesis química , Malaria Falciparum/tratamiento farmacológico , Femenino , Estructura MolecularRESUMEN
Pyruvate dehydrogenase kinase (PDK), which phosphorylates the pyruvate dehydrogenase complex, regulates glucose metabolism in skeletal muscle. PDK1, an isozyme whose expression is controlled by hypoxia-inducible factor-1α (HIF-1α), is thought to play a role in muscle adaptation to hypoxia. While transcriptional upregulation of PDK1 by HIF-1α is well characterised, mechanisms controlling proteolysis of PDK1 in skeletal muscle have not been thoroughly investigated. Proteasome inhibitor MG132 paradoxically reduced the abundance of PDK1 in human cancer cells and rat L6 myotubes, suggesting that MG132 might direct PDK1 towards autophagic degradation. The objectives of our current study were to determine (1) whether MG132 suppresses PDK1 levels in primary human myotubes, (2) whether chloroquine, an inhibitor of autophagy, prevents MG132-induced suppression of PDK1 in L6 myotubes, and (3) whether PYR-41, an inhibitor of ubiquitination, suppresses PDK1 in L6 myotubes. Using qPCR and/or immunoblotting, we found that despite markedly upregulating HIF-1α protein, MG132 did not alter the PDK1 expression in cultured primary human myotubes, while it suppressed both PDK1 mRNA and protein in L6 myotubes. The PDK1 levels in L6 myotubes were suppressed also during co-treatment with chloroquine and MG132. PYR-41 markedly increased the abundance of HIF-1α in primary human and L6 myotubes, while reducing the abundance of PDK1. In L6 myotubes treated with PYR-41, chloroquine increased the abundance of the epidermal growth factor receptor, but did not prevent the suppression of PDK1. Collectively, our results suggest that cultured myotubes degrade PDK1 via a pathway that cannot be inhibited by MG132, PYR-41, and/or chloroquine.
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Fibras Musculares Esqueléticas , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Animales , Humanos , Ratas , Células Cultivadas , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Leupeptinas/farmacología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Ubiquitina/metabolismoRESUMEN
BACKGROUND: Resistance to chemotherapy is a major problem in the treatment of patients with triple-negative breast cancer (TNBC). Preclinical data suggest that TNBC is dependent on proteasomes; however, clinical observations indicate that the efficacy of proteasome inhibitors in TNBC may be limited, suggesting the need for combination therapies. METHODS: We compared bortezomib and carfilzomib and their combinations with nelfinavir and lopinavir in TNBC cell lines and primary cells with regard to their cytotoxic activity, functional proteasome inhibition, and induction of the unfolded protein response (UPR). Furthermore, we evaluated the involvement of sXBP1, ABCB1, and ABCG2 in the cytotoxic activity of drug combinations. RESULTS: Carfilzomib, via proteasome ß5 + ß2 inhibition, is more cytotoxic in TNBC than bortezomib, which inhibits ß5 + ß1 proteasome subunits. The cytotoxicity of carfilzomib was significantly potentiated by nelfinavir or lopinavir. Carfilzomib with lopinavir induced endoplasmic reticulum stress and pro-apoptotic UPR through the accumulation of excess proteasomal substrate protein in TNBC in vitro. Moreover, lopinavir increased the intracellular availability of carfilzomib by inhibiting carfilzomib export from cells that express high levels and activity of ABCB1, but not ABCG2. CONCLUSION: Proteasome inhibition by carfilzomib combined with nelfinavir/lopinavir represents a potential treatment option for TNBC, warranting further investigation.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Bortezomib , Sinergismo Farmacológico , Inhibidores de la Proteasa del VIH , Lopinavir , Nelfinavir , Oligopéptidos , Neoplasias de la Mama Triple Negativas , Respuesta de Proteína Desplegada , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Oligopéptidos/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Nelfinavir/farmacología , Línea Celular Tumoral , Lopinavir/farmacología , Femenino , Bortezomib/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Respuesta de Proteína Desplegada/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacosRESUMEN
There is an urgent need to develop new, safer, and more effective drugs against Chagas disease (CD) as well as related kinetoplastid diseases. Targeting and inhibiting the Trypanosoma cruzi proteasome has emerged as a promising therapeutic approach in this context. To expand the chemical space for this class of inhibitors, we performed virtual screening campaigns with emphasis on shape-based similarity and ADMET prioritization. We describe the ideation and application of robustly validated shape queries for these campaigns, which furnished 44 compounds for biological evaluation. Five hit compounds demonstrated in vitro antitrypanosomal activity by potential inhibition of T. cruzi proteasome and notable chemical diversities, particularly, LCQFTC11. Structural insights were achieved by homology modeling, sequence/structure alignment, proteasome-species comparison, docking, molecular dynamics, and MMGBSA binding affinity estimations. These methods confirmed key interactions as well as the stability of LCQFTC11 at the ß4/ß5 subunits' binding site of the T. cruzi proteasome, consistent with known inhibitors. Our results warrant future assay confirmation of our hit as a T. cruzi proteasome inhibitor. Importantly, we also shed light into dynamic details for a proteasome inhibition mechanism that shall be further investigated. We expect to contribute to the development of viable CD drug candidates through such a relevant approach.
Asunto(s)
Simulación del Acoplamiento Molecular , Complejo de la Endopetidasa Proteasomal , Inhibidores de Proteasoma , Trypanosoma cruzi , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/química , Simulación de Dinámica Molecular , Tripanocidas/farmacología , Tripanocidas/química , Sitios de Unión , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Unión ProteicaRESUMEN
Canine urothelial carcinoma (cUC) is one of the most malignant tumors affecting dogs; however, its proliferative mechanism is yet to be fully elucidated. The ubiquitin-proteasome system (UPS) is an important metabolic pathway regulating protein degradation, and its dysfunction leads to apoptosis. We investigated the antitumor effect of the proteasome inhibitor bortezomib, which blocks the UPS. Bortezomib inhibited cell growth in cUC cell lines by inducing apoptosis in vitro. These findings suggest the potential of bortezomib as a novel therapeutic drug for dogs with cUC.
Asunto(s)
Antineoplásicos , Apoptosis , Bortezomib , Enfermedades de los Perros , Inhibidores de Proteasoma , Neoplasias de la Vejiga Urinaria , Animales , Perros , Bortezomib/farmacología , Bortezomib/uso terapéutico , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Enfermedades de los Perros/tratamiento farmacológico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/veterinaria , Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/veterinariaRESUMEN
Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Complejo de la Endopetidasa Proteasomal/metabolismo , Humanos , Especificidad por Sustrato , Unión Proteica , Proteolisis , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/química , Ubiquitina/metabolismo , AnimalesRESUMEN
Triptolide (TP) is a major active and toxic composition of the Chinese medicine Tripterygium wilfordii Hook. F. (TWHF), exhibiting various therapeutic bioactivities. Among the toxic effects, the hepatotoxicity of TP deserves serious attention. Previously, our research group proposed a new view of TP-related hepatotoxicity: hepatic hypersensitivity under lipopolysaccharide (LPS) stimulation. However, the mechanism of TP/LPS-induced hepatic hypersensitivity remains unclear. In this study, we investigated the mechanism underlying TP/LPS-induced hypersensitivity from the perspective of the inhibition of proteasome activity, activated endoplasmic reticulum stress (ERS)-related apoptosis, and the accumulation of reactive oxygen species (ROS). Our results showed that N-acetylcysteine (NAC), a common ROS inhibitor, decreased the expression of cleaved caspase-3 and cleaved PARP, which are associated with FLIP enhancement. Moreover, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was able to alleviate TP/LPS-induced hepatotoxicity by reducing ERS-related apoptosis protein expression (GRP78, p-eIF2α/eIF2α, ATF4, CHOP, cleaved caspase-3 and cleaved PARP) and ROS levels, with ATF4 being an indispensable mediator. In addition, the proteasome activity inhibitor MG-132 further aggravated ERS-related apoptosis, which indicated that the inhibition of proteasome activity also plays an important role in TP/LPS-related liver injuries. In summary, we propose that TP/LPS may upregulate the activation of ERS-associated apoptosis by inhibiting proteasome activity and enhancing ROS production through ATF4.
Asunto(s)
Acetilcisteína , Apoptosis , Diterpenos , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Compuestos Epoxi , Lipopolisacáridos , Fenantrenos , Complejo de la Endopetidasa Proteasomal , Inhibidores de Proteasoma , Especies Reactivas de Oxígeno , Fenantrenos/farmacología , Fenantrenos/toxicidad , Diterpenos/farmacología , Diterpenos/toxicidad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Apoptosis/efectos de los fármacos , Lipopolisacáridos/toxicidad , Compuestos Epoxi/toxicidad , Compuestos Epoxi/farmacología , Animales , Especies Reactivas de Oxígeno/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Acetilcisteína/farmacología , Factor de Transcripción Activador 4/metabolismo , Fenilbutiratos/farmacología , Ratones , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hígado/efectos de los fármacos , Hígado/patología , Hígado/metabolismo , Caspasa 3/metabolismo , Masculino , LeupeptinasRESUMEN
Glioblastoma (GB) remains a formidable challenge and requires new treatment strategies. The vital part of the Ubiquitin-proteasome system (UPS) in cellular regulation has positioned it as a potentially crucial target in GB treatment, given its dysregulation oncolines. The Ubiquitin-specific proteases (USPs) in the UPS system were considered due to the garden role in the cellular processes associated with oncolines and their vital function in the apoptotic process, cell cycle regulation, and autophagy. The article provides a comprehensive summary of the evidence base for targeting USPs as potential factors for neoplasm treatment. The review considers the participation of the UPS system in the development, resulting in the importance of p53, Rb, and NF-κB, and evaluates specific goals for therapeutic administration using midnight proteasomal inhibitors and small molecule antagonists of E1 and E2 enzymes. Despite the slowed rate of drug creation, recent therapeutic discoveries based on USP system dynamics hold promise for specialized therapies. The review concludes with an analysis of future wanderers and the feasible effects of targeting USPs on personalized GB therapies, which can improve patient hydration in this current and unattractive therapeutic landscape. The manuscript emphasizes the possibility of USP oncogene therapy as a promising alternative treatment line for GB. It stresses the direct creation of research on the medical effectiveness of the approach.
Asunto(s)
Glioblastoma , Proteasas Ubiquitina-Específicas , Humanos , Glioblastoma/patología , Glioblastoma/tratamiento farmacológico , Glioblastoma/enzimología , Glioblastoma/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Terapia Molecular Dirigida/métodos , Complejo de la Endopetidasa Proteasomal/metabolismo , Antineoplásicos/uso terapéutico , Animales , Inhibidores de Proteasoma/uso terapéutico , Inhibidores de Proteasoma/farmacologíaRESUMEN
Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.